The measurement of whole blood hemoglobin (Hb) is frequently performed using manual spectrophotometric methods and using semi-automated and automated hematology analyzers and methods and reagents used therein. Recent advances in high-throughput (e.g., up to about 100 samples per hour) hematology analyzers, e.g., the TECHNICON H.circle-solid..TM. series, including, but not limited to, the TECHNICON H.circle-solid.1.TM., H.circle-solid.2.TM. and H.circle-solid.3.TM. systems have led to the need for Hb methods that reach completion in about 30 seconds or less.
Cyanide-containing and cyanide-free Hb methods and reagents are used in automated Hb analyzers, such as the TECHNICON H.circle-solid..TM. series of instruments, to rapidly and quantitatively convert any carboxyhemoglobin (HbCO) in blood samples to a quantifiable reaction product, for example, in less than about 24 seconds. It has been found that Hb assays performed using either automated analyzers, for example, the TECHNICON H.circle-solid..TM. series of hematology analyzers, are not affected by the presence of up to 100% HbCO as a percentage of total blood hemoglobin. In addition, it has been found that both cyanide-containing and cyanide-free methods performed using automated TECHNICON H.circle-solid..TM. analyzers were comparable with respect to the performance parameters of linearity, precision and carryover. Further, these methods correlated well with the International Committee for Standardization of Hematology (ICSH) Manual Reference Hb Method on normal and abnormal blood samples, thus indicating acceptable accuracy (M. J. Malin et al., 1989, Am. J. Clin. Path., 92:286-294; M. J. Malin et al. 1992, Analyt. Chim. Acta, 262:67-77).
With regard to the analysis of abnormal blood samples which may contain aberrantly high levels of cells, particularly white blood cells, it may be difficult for current Hb determination methods and the reagents used therein to provide accurate and precise values. According to Wintrobe et al. (1981, Clinical Hematology, Lea and Febiger, Philadelphia, Pa., p. 208), a normal white blood cell count is on the order of about 3 to 10.times.10.sup.3 cells/.mu.l of blood. Thus, a moderately high normal count can be considered to be, for example, about 8.times.10.sup.3 cells/.mu.l, and an abnormally elevated white blood cell count can be considered to be about 10.times.10.sup.3 cells/.mu.l or greater.
U.S. Pat. No. 3,874,852 to T. E. Hammill discloses a reagent for the determination of leukocytes and hemoglobin in blood comprising a ferricyanide ion-free solution containing a quaternary ammonium ionic surfactant and an inorganic cyanide salt in which the pH of the reagent is approximately 9 and the pH of the final buffered solution used for analysis is 7.6 when employed with the commercial buffered blood diluent Isoton.RTM..
Cyanide-containing hemoglobin reagents having a reagent pH in the range of from 11.2 to 11.5 or 11.6 are commerically available from the assignee hereof. However, as described herein, unexpected problems in Hb determinations using the available Hb reagents having particular pH values were not solved until the present inventor's discovery of the causes of the problems and the development of reagent compositions and methods in accordance with the present invention.
Accordingly, needed in the art are methods and reagents which eliminate interference caused by moderate to high white blood cell counts and which yield acceptable imprecision in the determination of hemoglobin. Also needed are methods and reagents which do not suffer from system-to-system variation and which are capable of accurately quantifying Hb content in situations of both moderately to highly elevated white blood cell counts in normal or abnormal blood samples.
Another problem in the art arises when a blood sample remains mixed with a Hb reagent composition in a reaction mixture for some time prior to Hb analysis. Differences in reaction rates of Hb methods can depend on the mechanistic aspects of the blood in the reaction mixture with the Hb reagent. For example, a large increase in the reaction rate of an automated method compared with the ICSH method can depend on the disruption of the Hb molecule and the extraction of a ligated hemin derivative by surfactant micelles in the former method. By contrast, in the ICSH method, the integrity of the Hb molecular structure is maintained and the oxidation of heme iron and the subsequent ligation by cyanide occurs within the intact Hb structure (M. J. Malin et al., 1989, Am. J. Clin. Path., 92:286-294; M. J. Malin et al., 1992, Analyt. Chim. Acta, 262:67-77). For these reasons among others, current Hb determination reagents and methods used for particular automated instrumentation, such as the TECHNICON H.circle-solid..TM. series of analyzers, may not always perform optimally under conditions of high white blood cell counts. In such cases, new reagents need to be developed and used by those in the art to avoid measurement imprecision and variation in results among different instruments or different instruments in a series, such as the TECHNICON H.circle-solid..TM. analyzer series.
In view of the popular clinical utilization of whole blood Hb measurement and for the reasons presented hereinabove, those in the art are attuned both to maintaining the precision and accuracy of these spectrophotometric-based assays and to improving upon the analysis conditions and reagent compositions used in Hb determination methods. With respect to the reagent compositions for Hb analysis and measurement, ionic cyanide is still routinely used by those skilled in the art as a reagent component for Hb determination with effective, accurate and precise results. Needed in the art are methods and reagent compositions for Hb measurement and analysis that can be used with acceptable accuracy and precision in all types of hematology analyzers, for example, a cyanide-containing Hb method and reagent composition having superior performance capabilities in each of the TECHNICON H.circle-solid..TM. series of analyzers with little to no system-to-system variation. The reagent compositions should optimally be formulated so that all of the specified features and components of such reagents, including the specifications for pH, cyanide, total alkalinity, surfactant concentration, osmolality and surface tension, provide precise results for the analysis of both normal and abnormal blood samples, which may contain both normal and unusually high levels of white blood cells.